Vitamin-mitomycin conjugates

ABSTRACT

This invention relates to vitamin-mitomycin conjugates, to a method of using the conjugates to selectively eliminate a population of pathogenic cells in a host animal harboring the pathogenic cells, and to a method of preparation of the conjugates. The conjugate is of the general formula
 
B-L-X
 
wherein the group B is a vitamin, or an analog or a derivative thereof, that binds to a surface accessible vitamin receptor that is uniquely expressed, overexpressed, or preferentially expressed by a population of pathogenic cells, wherein the group L comprises a cleavable linker, and wherein the group X comprises a mitomycin compound, or an analog or a derivative thereof. An additional therapeutic agent, such as a chemotherapeutic agent, can be administered in combination with the conjugate.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a U.S. national counterpart application ofinternational application serial no. PCT/US03/14969 filed May 13, 2003,which claims the benefit of U.S. provisional application Ser. Nos.60/380,579 and 60/425,918 filed May 15, 2002, and Nov. 13, 2002,respectively.

FIELD OF THE INVENTION

This invention relates to compositions and methods for use in treatingdisease states characterized by the proliferation of pathogenic cellpopulations. More particularly, the invention relates tovitamin-mitomycin conjugates, to a method of using the conjugates toselectively eliminate a population of pathogenic cells in a host animal,and to a method of preparation of the conjugates.

BACKGROUND OF THE INVENTION

The mammalian immune system provides a means for the recognition andelimination of tumor cells, other pathogenic cells, and invading foreignpathogens. While the immune system normally provides a strong line ofdefense, there are many instances where cancer cells, other pathogeniccells, or infectious agents evade a host immune response and proliferateor persist with concomitant host pathogenicity. Chemotherapeutic agentsand radiation therapies have been developed to eliminate replicatingneoplasms. However, most, if not all, of the currently availablechemotherapeutic agents and radiation therapy regimens have adverse sideeffects because they work not only to destroy cancer cells, but theyalso affect normal host cells, such as cells of the hematopoieticsystem. The adverse side effects of the currently available anticancerdrugs highlight the need for the development of new therapies specificfor pathogenic cell populations and with reduced host toxicity.

Researchers have developed therapeutic protocols for destroying cancercells by targeting cytotoxic compounds to such cells. Many of theseprotocols utilize toxins conjugated to antibodies that bind to antigensunique to or overexpressed by cancer cells in an attempt to minimizedelivery of the toxin to normal cells. Using this approach certainimmunotoxins have been developed consisting of antibodies directed tospecific antigens on pathogenic cells, the antibodies being linked totoxins such as ricin, Pseudomonas exotoxin, Diptheria toxin, and tumornecrosis factor. These immunotoxins target tumor cells bearing thespecific antigens recognized by the antibody (Olsnes, S., Immunol.Today, 10, pp. 291-295, 1989; Melby, E. L., Cancer Res., 53(8), pp.1755-1760, 1993; Better, M. D., PCT Publication Number WO 91/07418,published May 30, 1991). Although the immunotoxins are directed tospecific antigens on pathogenic cells, the toxin component of thesecompounds may exhibit toxicity to normal host cells. The use of vitaminsto deliver chemotherapeutic agents to cells has also been described (seeU.S. Pat. No. 5,416,016).

Another approach for targeting populations of cancer cells or foreignpathogens in a host is to enhance the host immune response against thepathogenic cells to avoid the need for administration of compounds thatmay also exhibit independent host toxicity. One reported strategy forimmunotherapy is to bind antibodies, for example, genetically engineeredmultimeric antibodies, to the tumor cell surface to display the constantregion of the antibodies on the cell surface and thereby induce tumorcell killing by various immune-system mediated processes (De Vita, V.T., Biologic Therapy of Cancer, 2d ed. Philadelphia, Lippincott, 1995;Soulillou, J. P., U.S. Pat. No. 5,672,486). However, these approacheshave been complicated by the difficulties in defining tumor-specificantigens. Thus, there remains a significant need for effective therapieswith minimized host toxicity directed to the treatment of disease statescharacterized by the existence of pathogenic cell populations in theaffected host.

Mitomycins are natural products known to exhibit anti-tumor activity.Mitomycins can be produced by fermentation of Streptomyces caespitosus,and representive known mitomycins include mitomycin A, mitomycin B,mitomycin C, mitomycin D, mitomycin E, mitomycin F, and porfiromycin.The structures of these compounds are depicted by the following genericformula with substituents as shown in Table 1.

TABLE 1 Mitomycin X Y Z C-9 A OCH₃ CH₃ H β B OCH₃ H CH₃ α C NH₂ CH₃ H βD NH₂ H CH₃ α E NH₂ CH₃ CH₃ α F OCH₃ CH₃ CH₃ β J OCH₃ CH₃ CH₃ αPorfiromycin NH₂ CH₃ CH₃ β

Mitomycins are a class of cytotoxic drugs known as quinone-containingalkylating agents. Reduction of the quinone moiety results in theformation of bi-functional alkylating species that can form covalentbonds with a variety of cellular components including DNA. Theinteraction with DNA results in the formation of DNA crosslinks leadingto the induction of apoptosis and cell death, and this interaction isthought to be the most important contributor to the anti-tumor activityof mitomycin compounds.

SUMMARY OF THE INVENTION

The present invention relates to conjugates comprising a vitamin moietylinked to a mitomycin compound by a cleavable linker, to their use inthe treatment of disease states characterized by the proliferation of apathogenic cell population, and to a method of preparation of theconjugates. The vitamin-mitomycin conjugates in accordance with theinvention can be used to selectively eliminate a population ofpathogenic cells in an affected host. The selective elimination of thepathogenic cells is mediated by the binding of the vitamin moiety of thevitamin-mitomycin conjugate to a vitamin receptor, transporter, or othersurface-presented protein that specifically binds the targeting vitamin,and which is uniquely expressed, overexpressed, or preferentiallyexpressed by the pathogenic cells. The vitamin-mitomycin conjugates canbe internalized into the targeted cells upon binding of the vitaminmoiety to such a receptor, transporter, or surface-expressed protein,and, with the presence of a cleavable linker used to conjugate thevitamin moiety to the mitomycin compound, the vitamin moiety and themitomycin compound can dissociate intracellularly. The dissociatedmitomycin compound thereafter interacts with DNA, such as by formingcrosslinks with DNA, resulting in killing or inhibition of proliferationof the pathogenic cells.

Surface-expressed vitamin receptors, such as the high-affinity folatereceptor, are overexpressed, for example, on cancer cells. Epithelialcancers of the ovary, mammary gland, colon, lung, nose, throat, andbrain have all been reported to express elevated levels of the folatereceptor. In fact, greater than 90% of all human ovarian tumors areknown to express large amounts of this receptor. Accordingly, thepresent invention can be used for killing or inhibiting theproliferation of a variety of tumor cell types, and of other types ofpathogenic cells that overexpress vitamin receptors.

Although mitomycins are known to exhibit excellent anti-tumor activity,mitomycins also exhibit cytoxicity towards leukocytes in the host animaltreated with these compounds. In an effort to increase the anti-tumoractivity of mitomycins and/or to decrease the undesired toxicity ofthese compounds, derivatives of mitomycins have been prepared whichcontain a variety of modifications at the C-7-amino group in theaminomitosane skeleton. Among the known mitomycin derivatives withreduced toxicity are unsymmetrical dialkyl disulfides containing C-7substituents of the formula RSS(CH₂)₂NH— in which i) R is an alkyl group(see European Patent Application No. 0116208A1 and Japanese PatentApplication No. 175493/84), ii) R contains an aromatic ring (seeEuropean Patent Application Nos. 0116208A1 and 0163550A2, JapanesePatent Application No. 255789/85, and U.S. Pat. No. 4,866,180), and iii)R is structurally related to amino acid or peptide fragments (seeEuropean Patent Application No. 0163550A2, Japanese Patent ApplicationNo. 255789/85, and U.S. Pat. No. 4,691,024).

All known methods for the preparation of such unsymmetrical dialkyldisulfides are based on a transthiolation of unsymmetricalheteroaryl-alkyl disulfides with alkyl thiol. Notably, the most commonheteroarylthio leaving groups are 2-thiopyridyl (see WO 88/01622 andEuropean App. Publication No. 0116208A1) and 3-nitro-2-thiopyridine (seeU.S. Pat. No. 4,691,024). The driving force for this cleavage reactionis the excellent leaving group properties of the heteroarylthio moiety.The reaction is as follows:

The vitamin-mitomycin conjugates in accordance with the presentinvention can be prepared using such transthiolation methods or, forexample, by a novel process for the preparation of unsymmetrical dialkyldisulfides that is based on the thiophilicity of thiosulfonate reagents.The synthetic scheme for the preparation of the thiosulfonate reagents,mitomycin A, and for the synthesis of a vitamin derivative (e.g.,cysteine-terminated folate) for use in this preparation process is shownin scheme 1 below (Fmoc=9-fluorenylmethyloxycarbonyl;Boc=tert-butyloxycarbonyl; Dap=diaminopropionic acid;DMF=dimethylformamide; DIPEA=diisopropylethylamine;DMSO=dimethylsulfoxide; TFAA=trifluoroacetic acid;PyBOP=benzotriazole-1-yl-oxy-tris-(pyrrolidinophosphoniumhexafluoro-phosphate)).

Exemplary synthetic schemes for the synthesis of the presentvitamin-mitomycin conjugates as unsymmetrical dialkylsulfides usingthiosulfonates are presented as schemes 2 and 3 below. In thedisulfide-linked conjugates, one of the alkyl moieties is attached tothe C-7-amino group in the mitosane skeleton, and the other iscovalently attached via a divalent linker or directly to a vitaminmolecule, or a vitamin receptor binding analog or derivative thereof.

In both schemes 2 and 3, a commercially available amine 1, containing athiolsulfonate group, was first reacted with the vinylogous methyl esterat C-7 in the quinone moiety of the mitosane derivative 2. Second, theresulting thiolsulfonate reagent 3 is used for sulfonylation of thiols,such as that found on pteroyl-glutamyl-cysteine 4, onPte-Glu-Asp-Arg-Asp-Cys-OH 6, or Pte-Glu-Arg-Cys-Ala-Gly-OH 8, which areall folic acid derivatives. The instantaneous disulfide formationproduces a vitamin-mitomycin conjugate, for example, apteroyl-Glu-Cys-S-mitomycin C conjugate 5, aPte-Glu-Asp-Arg-Asp-Cys-S-mitomycin C—OH conjugate 7, or aPte-Glu-Arg-Cys-S-mitomycin C-Ala-Gly-O conjugate 9, in almostquantitative yield.

In embodiments of the present invention where the disulfide linkage isan unsymmetrical dialkyl disulfide, such as an unsymmetrical dialkyldisulfide prepared by the above-described procedure, thevitamin-mitomycin conjugates selectively eliminate the pathogenic cells,and have reduced toxicity towards normal cells. In embodiments where thevitamin moiety is conjugated to a mitomycin compound by such a disulfidelinkage, the vitamin moiety and mitomycin compound can dissociate underthe reducing conditions that exist intracellularly.

In one embodiment is provided a conjugate of the general formulaB-L-Xwherein the group B is a vitamin, or an analog or a derivative thereof,that binds to a surface accessible vitamin receptor that is uniquelyexpressed, overexpressed, or preferentially expressed by a population ofpathogenic cells, wherein the group L comprises a cleavable linker, andwherein the group X comprises a mitomycin compound or an analog or aderivative thereof.

In another embodiment is provided a method of selectively eliminating apopulation of pathogenic cells in a host animal harboring the populationof cells wherein the members of the cell population have a surfaceaccessible binding site for a vitamin. The method comprises the steps ofadministering to the host a conjugate of the general formulaB-L-Xwherein the group B is a vitamin, or an analog or a derivative thereof,that binds to a surface accessible vitamin receptor that is uniquelyexpressed, overexpressed, or preferentially expressed by the populationof pathogenic cells, wherein the group L comprises a cleavable linker,and wherein the group X comprises a mitomycin compound or an analog or aderivative thereof, and selectively eliminating the population ofpathogenic cells.

In an alternate embodiment, the method can further comprise the step ofadministering to the host animal a chemotherapeutic agent such aspaclitaxel.

In yet another embodiment is provided a pharmaceutical compositioncomprising a conjugate of the general formulaB-L-Xwherein the group B is a vitamin, or an analog or a derivative thereof,that binds to a surface accessible vitamin receptor that is uniquelyexpressed, overexpressed, or preferentially expressed by a population ofpathogenic cells, wherein the group L comprises a cleavable linker, andwherein the group X comprises a mitomycin compound or an analog or aderivative thereof, and a pharmaceutically acceptable carrier therefor.

In another embodiment, the pharmaceutical composition can furthercomprise a chemotherapeutic agent such as paclitaxel.

In still another embodiment is provided a method of preparing abiologically active conjugate of the formulaB-L-X

wherein B is a vitamin or a vitamin-receptor-binding analog orderivative thereof;

X comprises a mitomycin compound or an analog or derivative thereof;

and L is a divalent linker comprising a disulfide bond, the methodcomprising the steps of forming a thiosulfonate intermediate of theformula B-(L′)_(n)SSO₂R or an intermediate of the formulaX-(L′)_(n)SSO₂R

and reacting the thiosulfonate intermediate with a compound of theformula X-(L′)_(n′)-SH or B-(L″)_(n′)-SH, respectively, wherein L′ andL″ are, independently, divalent linkers through which the thiol group SHis covalently bonded to B and X, respectively;

n and n′ are 1 or 0; and

R is alkyl, substituted alkyl, aryl, heteroaryl or substituted aryl orheteroaryl.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a comparison between the cytotoxicities of EC72 (see scheme1; closed triangles) and mitomycin C (closed squares). The cytotoxicityof EC72 in the presence of excess free folate (closed circles) is alsoshown.

FIG. 2 shows the percentage survival of M109 tumor-bearing mice injectedwith PBS (control; closed squares), mitomycin C (open circles), or EC72(closed circles).

FIG. 3 shows the percentage survival of M109 tumor-bearing mice injectedwith PBS (control; closed triangles), mitomycin C (closed squares), EC72(closed circles), or EC72 plus excess free folate (closed diamonds).

FIG. 4 shows tumor size in mice implanted with M109 cells to formsubcutaneous tumors and treated with PBS (closed squares) or EC72(closed circles).

FIG. 5 shows 1D NMR of a pteroyl-Glu-Cys-S-mitomycin C conjugate(compound 5; see scheme 2), a Pte-Glu-Asp-Arg-Asp-Cys-S-mitomycin C—OHconjugate (compound 7; see scheme 2), and a Pte-Glu-Arg-Cys-S-mitomycinC-Ala-Gly-O conjugate (compound 9; see scheme 3).

FIG. 6 shows 2D NMR of a pteroyl-Glu-Cys-S-mitomycin C conjugate(compound 5; see scheme 2), a Pte-Glu-Asp-Arg-Asp-Cys-S-mitomycin C—OHconjugate (compound 7; see scheme 2), and a Pte-Glu-Arg-Cys-S-mitomycinC-Ala-Gly-O conjugate (compound 9; see scheme 3).

FIG. 7 shows tumor size in mice implanted with M109 cells to formsubcutaneous tumors and treated with PBS (closed squares) or taxol (20mg/kg; open circles), or taxol plus EC72 (closed circles).

FIG. 8 shows tumor size in mice implanted with M109 cells to formsubcutaneous tumors and treated with PBS (closed squares) or taxol (15mg/kg; open circles), or taxol plus EC72 (closed circles).

FIG. 9 shows ³H-folic acid binding to KB cells in the presence ofincreasing concentrations of free folic acid (lower curve) or EC72(upper curve).

FIG. 10 shows the inhibition of KB cell DNA synthesis by increasingconcentrations of EC72 (closed circles) or EC72 in the presence of 0.1mM free folate (open circles).

FIG. 11 shows the percentage survival of M109 tumor-bearing miceinjected with PBS (control; closed squares) or increasing concentrationsof EC72 (closed diamonds=100 nmol/kg of EC72, closed triangles=400nmol/kg of EC72, and closed circles=1800 nmol/kg of EC72).

FIG. 12 shows tumor size in mice implanted with folate receptor-negative4T-1 cells to form subcutaneous tumors and treatment with PBS (closedsquares) or EC72 (closed circles).

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to conjugates comprising a vitamin moietylinked to a mitomycin compound by a cleavable linker. Thevitamin-mitomycin conjugates in accordance with the invention can beused to selectively eliminate a population of pathogenic cells in anaffected host. The selective elimination of the pathogenic cells ismediated by the binding of the vitamin moiety of the vitamin-mitomycinconjugate to a vitamin receptor, transporter, or other surface-presentedprotein that specifically binds vitamins or vitamin analogs and which isuniquely expressed, overexpressed, or preferentially expressed by thepathogenic cells. A surface-presented protein uniquely expressed,overexpressed, or preferentially expressed by the pathogenic cells ispreferably a receptor not present or present at lower concentrations onnon-pathogenic cells providing a means for selective elimination of thepathogenic cells.

The vitamin-mitomycin conjugates are internalized upon binding of thevitamin moiety to such a receptor, transporter, or surface-expressedprotein and the vitamin moiety and the mitomycin compound can dissociateintracellularly upon cleavage of a cleavable linker used to covalentlylink the vitamin moiety to the mitomycin compound. The cleavable linkercan be, for example, a disulfide linkage which results in reducedtoxicity of the vitamin-mitomycin conjugate towards normal cells. Inembodiments where the vitamin moiety is conjugated to a mitomycincompound by a disulfide linkage, the vitamin moiety and mitomycincompound can dissociate under the reducing conditions that existintracellularly. Upon its dissociation from the vitamin moiety, themitomycin compound can interact with DNA, such as by forming crosslinkswith DNA, resulting in killing or inhibiting the proliferation of thepathogenic cells.

In an alternative embodiment, the vitamin moiety of the conjugate canbind to the pathogenic cell placing the mitomycin compound in closeassociation with the cell surface. The drug can then be released bycleavage of the disulfide linkage, for example, by a protein disulfideisomerase. The mitomycin compound can be taken up by the pathogenic cellto which the vitamin-mitomycin conjugate is bound, or the mitomycincompound can be taken up by another pathogenic cell in close proximitythereto to interact with the cell's DNA and kill or inhibitproliferation of the pathogenic cell. Alternatively, the drug could bereleased by a protein disulfide isomerase inside the cell where thereleasable linker is a disulfide group.

In another embodiment, or in combination with the above-describedembodiments, the vitamin-mitomycin conjugates can act through amechanism independent of cellular vitamin receptors. For example, theconjugates can bind to soluble vitamin receptors present in the serum orto serum proteins, such as albumin, resulting in prolonged circulationof the conjugates relative to unconjugated mitomycin, and in increasedactivity of the conjugates towards the pathogenic cell populationrelative to unconjugated mitomycin.

The vitamin-mitomycin conjugates in accordance with the invention areutilized to selectively eliminate a population of pathogenic cells in ahost animal harboring the population of pathogenic cells. The inventionis applicable to populations of pathogenic cells that cause a variety ofpathologies including cancer, diseases mediated by activatedmacrophages, and diseases mediated by any other type of pathogenic cellsthat overexpress vitamin receptors or receptors that bind analogs orderivatives of vitamins. Thus, the population of pathogenic cells can bea cancer cell population that is tumorigenic, including benign tumorsand malignant tumors, or it can be non-tumorigenic. The cancer cellpopulation can arise spontaneously or by such processes as mutationspresent in the germline of the host animal or somatic mutations, or itcan be chemically-, virally-, or radiation-induced. The invention can beutilized to treat such cancers as carcinomas, sarcomas, lymphomas,Hodgekin's disease, melanomas, mesotheliomas, Burkitt's lymphoma,nasopharyngeal carcinomas, leukemias, and myelomas. The cancer cellpopulation can include, but is not limited to, oral, thyroid, endocrine,skin, gastric, esophageal, laryngeal, pancreatic, colon, bladder, bone,ovarian, cervical, uterine, breast, testicular, prostate, rectal,kidney, liver, and lung cancers.

In embodiments where the pathogenic cell population is a cancer cellpopulation, the effect of conjugate administration is a therapeuticresponse measured by reduction or elimination of tumor mass, maintenanceof tumor mass, or of inhibition of tumor cell proliferation. In the caseof a tumor, the elimination can be an elimination of cells of theprimary tumor or of cells that have metastasized or are in the processof dissociating from the primary tumor. A prophylactic treatment withthe vitamin-mitomycin conjugate to prevent return of a tumor after itsremoval by any therapeutic approach including surgical removal of thetumor, radiation therapy, chemotherapy, or biological therapy is alsocontemplated in accordance with this invention. The prophylactictreatment can be an initial treatment with the vitamin-mitomycinconjugate, such as treatment in a multiple dose daily regimen, and/orcan be an additional treatment or series of treatments after an intervalof days or months following the initial treatments(s). Accordingly,elimination of the pathogenic cell population includes elimination ofcells, inhibition of proliferation of pathogenic cells, maintenance oftumor mass, or a prophylactic treatment that prevents return ofpathogenic cells.

The method of the present invention can be used for both human clinicalmedicine and veterinary applications. Thus, the host animal harboringthe population of pathogenic cells and treated with vitamin-mitomycinconjugates can be human or, in the case of veterinary applications, canbe a laboratory, agricultural, domestic, or wild animal. The presentinvention can be applied to host animals including, but not limited to,humans, laboratory animals such rodents (e.g., mice, rats, hamsters,etc.), rabbits, monkeys, chimpanzees, domestic animals such as dogs,cats, and rabbits, agricultural animals such as cows, horses, pigs,sheep, goats, and wild animals in captivity such as bears, pandas,lions, tigers, leopards, elephants, zebras, giraffes, gorillas,dolphins, and whales.

In accordance with the present invention, the vitamin-mitomycinconjugates can be formed from a wide variety of vitamins orreceptor-binding vitamin analogs/derivatives and mitomycins. Thevitamin-mitomycin conjugates are capable of selectively targeting apopulation of pathogenic cells in the host animal due to preferentialexpression of a receptor for the vitamin, accessible for vitaminbinding, on the pathogenic cells. Acceptable vitamin moieties includeniacin, pantothenic acid, folic acid, riboflavin, thiamine, biotin,vitamin B₁₂, and the lipid soluble vitamins A, D, E and K. Thesevitamins, and their receptor-binding analogs and derivatives, constitutethe targeting entity that can be coupled with mitomycins by a cleavablelinker to form the vitamin-mitomycin conjugates for use in accordancewith the invention. Preferred vitamin moieties include folic acid,biotin, riboflavin, thiamine, vitamin B₁₂, and receptor-binding analogsand derivatives of these vitamin molecules, and other related vitaminreceptor-binding molecules (see U.S. Pat. No. 5,688,488, incorporatedherein by reference). Exemplary of a vitamin analog is a folate analogcontaining a glutamic acid residue in the D configuration (folic acidnormally contains one glutamic acid in the L configuration linked topteroic acid). The mitomycin compound can be any of the mitomycins ormitomycin-related compounds shown in Table 1 including mitomycin A,mitomycin B, mitomycin C, mitomycin D, mitomycin E, mitomycin F,mitomycin J, and porfiromycin, or analogs or derivatives thereof.

The binding site for the vitamin can include receptors for any vitaminmolecule, or a derivative or analog thereof, capable of specificallybinding to a receptor wherein the receptor or other protein is uniquelyexpressed, overexpressed, or preferentially expressed by a population ofpathogenic cells. A surface-presented protein uniquely expressed,overexpressed, or preferentially expressed by the pathogenic cells istypically a receptor that is either not present or present at lowerconcentrations on non-pathogenic cells providing a means for selectiveelimination of the pathogenic cells. In one embodiment, ligands that canbe used in the conjugates of the present invention include those thatbind to receptors expressed specifically on activated macrophages, suchas the folate receptor, which binds folate or an analog or derivativethereof.

In accordance with the invention the vitamin-mitomycin conjugates arecapable of high affinity binding to receptors on cancer cells or otherpathogenic cells. The high affinity binding can be inherent to thevitamin moiety or the binding affinity can be enhanced by the use of achemically modified vitamin (i.e., an analog or a derivative) or by theparticular chemical linkage between the vitamin and the mitomycincompound that is present in the conjugate.

The linker can be any biocompatible cleavable linker, such as a linkersusceptible to cleavage under the reducing conditions present in cells,an acid-labile linker, or an enzyme-labile linker. Typically, the linkercomprises about 1 to about 30 carbon atoms, more typically about 2 toabout 20 carbon atoms. Lower molecular weight linkers (i.e., thosehaving an approximate molecular weight of about 30 to about 300) aretypically employed.

Generally, any manner of forming a complex between the linker and thevitamin, or vitamin receptor binding derivative or analog, and betweenthe linker and the mitomycin can be utilized in accordance with thepresent invention. The complex can be formed by direct conjugation ofthe cleavable linker with the vitamin and the mitomycin, for example,through hydrogen, ionic, or covalent bonds. Covalent bonding of thevitamin, or vitamin receptor binding derivative or analog, and themitomycin with the linker can occur, for example, through the formationof amide, ester, disulfide, or imino bonds between acid, aldehyde,hydroxy, amino, sulfhydryl, or hydrazo groups. Also, in accordance withthis invention the linker can comprise an indirect means for associatingthe vitamin with the mitomycin, such as by connection throughintermediary linkers, spacer arms, or bridging molecules. Both directand indirect means for association should not prevent the binding of thevitamin, or vitamin receptor binding derivative or analog, to thevitamin receptor on the cell membrane for operation of the method of thepresent invention.

The invention also provides a method of preparing a biologically activeconjugate of the formulaB-L-X

wherein B is a vitamin or a vitamin-receptor-binding analog orderivative thereof;

X comprises a mitomycin compound or an analog or derivative thereof;

and L is a divalent linker comprising a disulfide bond, said methodcomprising the steps of forming a thiosulfonate intermediate of theformula B-(L′)_(n)SSO₂R or an intermediate of the formulaX-(L′)_(n)SSO₂R.

and reacting said thiosulfonate intermediate with a compound of theformula X-(L′)_(n′)-SH or B-(L″)_(n′)-SH, respectively, wherein L′ andL″ are, independently, divalent linkers through which the thiol group SHis covalently bonded to B and X, respectively;

n and n′ are 1 or 0; and

R is alkyl, substituted alkyl, aryl, heteroaryl or substituted aryl orheteroaryl.

The nature of the linking groups L′ and L″ in the intermediates are notcritical except that the linkers are preferably cleavable; nor is thenature of the group R on the thiosulfonate critical to the preparationof the vitamin conjugates. Precursors to such linking groups aretypically selected to have either nucleophilic or electrophilicfunctional groups, or both, optionally in a protected form with areadily cleavable protecting group to facilitate their use in synthesisof the intermediate species. The linking group -L- in the conjugate, byvirtue of the synthetic preparative procedure, can be represented as acomposite of the intermediate linking group L′ and L″ by the formula-(L′)_(n)-S—S-(L″)_(n′)-. In the case of -L′-, the entity X-(L′)_(n)-SHshould retain cytotoxic activity.

The invention is also directed to pharmaceutical compositions comprisingan amount of a vitamin-mitomycin conjugate effective to eliminate apopulation of pathogenic cells in a host animal when administered in oneor more doses. The vitamin-mitomycin conjugate is preferablyadministered to the host animal parenterally, e.g., intradermally,subcutaneously, intramuscularly, intraperitoneally, intravenously, orintrathecally. Alternatively, the conjugate can be administered to thehost animal by other medically useful processes, and any effective doseand suitable therapeutic dosage form, including prolonged release dosageforms, can be used. The method of the present invention can be used incombination with surgical removal of a tumor, radiation therapy,chemotherapy, or biological therapies such as other immunotherapiesincluding, but not limited to, monoclonal antibody therapy, treatmentwith immunomodulatory agents, adoptive transfer of immune effectorcells, treatment with hematopoietic growth factors, cytokines andvaccination.

Examples of parenteral dosage forms include aqueous solutions of theactive agent, in an isotonic saline, 5% glucose or other well-knownpharmaceutically acceptable liquid carriers such as liquid alcohols,glycols, esters, and amides. The parenteral dosage form in accordancewith this invention can be in the form of a reconstitutable lyophilizatecomprising the dose of the vitamin-mitomycin conjugate. In one preferredaspect of the present embodiment, any of a number of prolonged releasedosage forms known in the art can be administered such as, for example,the biodegradable carbohydrate matrices described in U.S. Pat. Nos.4,713,249; 5,266,333; and 5,417,982, the disclosures of which areincorporated herein by reference, or, alternatively, a slow pump (e.g.,an osmotic pump) can be used.

At least one additional composition comprising a therapeutic factor canbe administered to the host in combination or as an adjuvant to theabove-detailed methodology, to enhance the vitamin-mitomycin mediatedelimination of the population of pathogenic cells, or more than oneadditional therapeutic factor can be administered. The therapeuticfactor(s) can be selected from a compound capable of stimulating anendogenous immune response, a chemotherapeutic agent, or anothertherapeutic factor capable of complementing the efficacy of theadministered vitamin-mitomycin complex. The method of the invention canbe performed by administering to the host, in addition to theabove-described conjugates, compounds or compositions capable ofstimulating an endogenous immune response including, but not limited to,cytokines or immune cell growth factors such as interleukins 1-18, stemcell factor, basic FGF, EGP, G-CSF, GM-CSF, FLK-2 ligand, HILDA, MIP-1α,TGF α, TGF β, M-CSF, IFN α, IFN β, IFN γ, soluble CD23, LIF, andcombinations thereof.

Therapeutically effective combinations of these factors can be used. Inone embodiment, for example, therapeutically effective amounts of IL-2,for example, in amounts ranging from about 5000 IU/dose/day to about500,000 IU/dose/day in a multiple dose daily regimen, or, for example,in amounts ranging from about 7500 IU/dose/day to about 150,000IU/dose/day in a multiple dose daily regimen, can be used along with thevitamin-mitomycin conjugates to eliminate pathogenic cells in a hostanimal harboring such a population of cells. Alternatively, IL-2 can beused in combination with IFN-α where IL-2 can be used, for example, inamounts ranging from 0.1 MIU/m²/dose/day to about 15 MIU/m²/dose/day ina multiple dose daily regimen, and IFN-α, for example, can be used inamounts ranging from about 0.1 MIU/m²/dose/day to about 7.5MIU/m²/dose/day in a multiple dose daily regimen, along with theconjugates to eliminate or neutralize pathogenic cells in a host animalharboring the pathogenic cells (MIU=million international units;m²=approximate body surface area of an average human). In anotherembodiment IL-12 and IFN-α are used in therapeutically effectiveamounts, and in yet another embodiment OL-15 and IFN-α are used intherapeutically effective amounts. In an alternate embodiment IL-2,IFN-α or IFN-γ, and GM-CSF are used in combination. The invention alsocontemplates the use of any other effective combination of cytokinesincluding combinations of other interleukins and interferons and colonystimulating factors.

Chemotherapeutic agents, which are cytotoxic themselves, can work toenhance tumor permeability, can inhibit tumor growth or tumor cellproliferation, and the like, are also suitable for use in the method ofthe invention in combination with vitamin-mitomycin conjugates. Suchchemotherapeutic agents include adrenocorticoids, alkylating agents,antiandrogens, antiestrogens, androgens, estrogens, antimetabolites suchas cytosine arabinoside, purine analogs, pyrimidine analogs, andmethotrexate, actinomycin D, gemcitabine, busulfan, carboplatin,chlorambucil, cisplatin and other platinum compounds, trimethoprim,dicloxacillin, daunorubicin, doxorubicin, epirubicin, mitoxantrone,topotecan, etoposide, tamoxiphen, TAXOL® (i.e., paclitaxel),cyclophosphamide, cyclosporin, plant alkaloids, prednisone, hydroxyurea,teniposide, bleomycin, digoxin, nitrogen mustards, nitrosureas,vincristine, vinblastine, mitomycin C, inflammatory and proinflammatoryagents, and any other art-recognized chemotherapeutic agent.

The additional therapeutic factor can be administered to the host animalprior to, after, or at the same time as the vitamin-mitomycin conjugateand the therapeutic factor can be administered as part of the samecomposition containing the conjugate or as part of a differentcomposition than the vitamin-mitomycin conjugate. Any such therapeuticcomposition containing the therapeutic factor at a therapeuticallyeffective dose can be used in the present invention includingcompositions containing multiple therapeutic factors.

In one embodiment, the additional therapeutic factor is TAXOL® (i.e.,paclitaxel; sold by Bristol Myers Squibb Company under the trademarkTAXOL® and by Ivax Corporation under the trademark ONXOL™). Inaccordance with this invention, TAXOL®, ONXOL™, any other generic formof TAXOL®, or any related compound can be used in combination with thevitamin-mitomycin conjugates. TAXOL®, generic forms of TAXOL®, orrelated compounds can be administered, for example, to patients at dosesof about 10 to about 500 mg/square meter, about 50 to about 400mg/square meter, about 100 to about 300 mg/square meter, or about 100 toabout 200 mg/square meter, but any effective dose in combination withthe vitamin-mitomycin conjugates can be used.

Additionally, any effective regimen for administering TAXOL®, genericforms of TAXOL®, or a related compound can be used. For example, TAXOL®or generic or related compounds can be administered over 3 hours onceevery 3 weeks for a total of 18 weeks, but any other effective regimenis contemplated in accordance with this invention. TAXOL® or generic orrelated compounds can be administered by any effective route such asorally or parenterally, e.g., intradermally, subcutaneously,intramuscularly, intraperitoneally, intravenously, or intrathecally.

More than one type of drug delivery conjugate can be used. For example,the host animal can be treated with conjugates with different vitamins(e.g., folate-mitomycin conjugates and vitamin B₁₂-mitomycin conjugates)in a co-dosing protocol. In other embodiments, the host animal can betreated with conjugates comprising various vitamins linked to variousmitomycins. For example, the host animal could be treated with afolate-mitomycin C and a folate-mitomycin A conjugate, or with afolate-mitomycin C conjugate and a vitamin B₁₂-mitomycin A conjugate.Furthermore, drug delivery conjugates with the same or differentvitamins and the same or different mitomycins comprising multiplevitamins and multiple mitomycins as part of the same drug deliveryconjugate could be used.

The unitary daily dosage of the vitamin-mitomycin conjugate can varysignificantly depending on the host condition, the disease state beingtreated, the molecular weight of the conjugate, its route ofadministration and tissue distribution, and the possibility of co-usageof other therapeutic treatments such as radiation therapy. The effectiveamount to be administered to a patient is based on body surface area,patient weight, and physician assessment of patient condition. Aneffective dose can range from about 1 ng/kg to about 1 mg/kg, morepreferably from about 1 μg/kg to about 500 μg/kg, and most preferablyfrom about 1 μg/kg to about 100 μg/kg.

Any effective regimen for administering the vitamin-mitomycin conjugatecan be used. For example, the vitamin-mitomycin conjugate can beadministered as single doses, or it can be divided and administered as amultiple-dose daily regimen. Further, a staggered regimen, for example,one to three days per week can be used as an alternative to dailytreatment, and for the purpose of defining this invention suchintermittent or staggered daily regimen is considered to be equivalentto every day treatment and within the scope of this invention. In oneembodiment of the invention the host is treated with multiple injectionsof the vitamin-mitomycin conjugate to eliminate the population ofpathogenic cells. In one embodiment, the host is injected multiple times(preferably about 2 up to about 50 times) with the vitamin-mitomycinconjugate, for example, at 12-72 hour intervals or at 48-72 hourintervals. Additional injections of the vitamin-mitomycin conjugate canbe administered to the patient at an interval of days or months afterthe initial injections(s) and the additional injections preventrecurrence of the disease state caused by the pathogenic cells. Anyadditional therapeutic factor, such as a chemotherapeutic agent (e.g.,paclitaxel), can also be administered after initial injections toprevent recurrence of disease.

In one embodiment, ligands that can be used in the conjugates of thepresent invention include those that bind to receptors expressedspecifically on activated macrophages, such as the folate receptor whichbinds folate, or an analog or derivative thereof. The folate-mitomycinconjugates can be used to kill or suppress the activity of activatedmacrophages that cause disease states in the host. Such macrophagetargeting conjugates, when administered to a patient suffering from anactivated macrophage-mediated disease state, work to concentrate andassociate the conjugated mitomycin in the population of activatedmacrophages to kill the activated macrophages or suppress macrophagefunction. Elimination or deactivation of the activated macrophagepopulation works to stop or reduce the activated macrophage-mediatedpathogenesis characteristic of the disease state being treated.Exemplary of diseases known to be mediated by activated macrophagesinclude rheumatoid arthritis, ulcerative colitis, Crohn's disease,psoriasis, osteomyelitis, multiple sclerosis, atherosclerosis, pulmonaryfibrosis, sarcoidosis, systemic sclerosis, organ transplant rejection(GVHD) and chronic inflammations. Conjugate administration is typicallycontinued until symptoms of the disease state are reduced or eliminated,and the conjugate can be administered in combination with any additionaltherapeutic factor, such as a chemotherapeutic agent (e.g., paclitaxel).

The conjugates administered in accordance with the methods of thisinvention are preferably administered parenterally to the animal orpatient suffering from the disease state, for example, intradermally,subcutaneously, intramuscularly, intraperitoneally, or intravenously incombination with a pharmaceutically acceptable carrier. Alternatively,the conjugates can be administered to the animal or patient by othermedically useful procedures and effective doses can be administered instandard or prolonged release dosage forms. The therapeutic method ofthe present invention can be used alone or in combination with othertherapeutic methods recognized for treatment of macrophage mediateddisease states.

EXAMPLE 1 Preparation of Compounds 5, 7, and 9

Compounds 5, 7, and 9 (see schemes 2 and 3) were prepared as follows.First, compounds 1 and 2 (forty-five μmoles of each) were mixed withstirring under argon with 1 mL of anhydrous methanol. After stirring for20 hours, both starting materials disappeared as determined by TLC(silica gel; CHCl₃/MeOH=9/1). To prepare compound 5, in a separate flask1.0 mL of deionized water was added to forty-two μmoles of compound 4and the solution was purged with argon. To this solution was added 0.1 NNaHCO₃ until the pH was adjusted to 7, and this solution was mixed withthe first reaction solution described above (prepared by mixingcompounds 1 and 2). The conjugation reaction was completed in 30minutes. The methanol was evaporated i.vac. and the conjugate waspurified on preparative HPLC (Prep Novapak HR C18 19×300 mM column;mobile phase (A)-1.0 mM phosphate buffer, pH=6; organic phase(13)-acetonitrile; conditions-gradient from 99% A and 1% B to 50% A and50% B in 30 minutes, flow rate=15 mL/minute). Compounds 7 and 9 wereprepared using the same protocol except that compounds 6 and 8,respectively, were used in the second solution in place of compound 4.Compounds 5, 7, and 9 were identified by 1D (FIG. 5) and 2D NMR (FIG. 6)and MS (ES).

EXAMPLE 2 Cytotoxicity of EC72 and Mitomycin C

A compound denominated as EC72 (see scheme 1) was evaluated using an invitro cytotoxicity assay that predicts the ability of the drug toinhibit the growth of folate receptor-positive KB cells. This compoundcomprises a folate analog linked to mitomycin C prepared according tothe protocol depicted in scheme 2. The KB cells were exposed for 15minutes at 37° C. to the indicated concentrations of EC72, freemitomyicn C (drug #1), or to EC72 and at least a 100-fold excess offolic acid. The cells were then rinsed once with fresh culture mediumand incubated in fresh culture medium for 72 hours at 37° C. Cellviability was assessed using a bromodeoxyuridine-based ELISA assay.

When compared directly, the cytotoxicity of EC72 was equal to that offree mitomycin C (see FIG. 1). The cytotoxicity of EC72 was blocked inthe presence of excess free folic acid, indicating that the observedcell killing was mediated by folate binding to its receptor.Importantly, this result also suggests that normal tissues that expresslittle to no folate receptor should not be affected by the EC72conjugate.

EXAMPLE 3 Survival of Tumor-Bearing Mice Treated with EC72

The effectiveness of EC72 for promoting survival of tumor-bearing micewas evaluated in vivo using M109 tumor-bearing Balb/c mice. There weretwo goals of this study: i) to determine if daily intraperitonealtreatment with EC72 could prolong the lives of folate receptor-positivetumor-bearing mice beyond what mitomycin C could do when tested under anidentical dosing regimen, and ii) to examine the pathological effects ofEC72 treatment on normal tissues, including kidney tissue which isfolate receptor-positive tissue. Although most normal tissues containvery low levels of folate receptor, the proximal tubules of the kidneyexpress appreciable numbers of folate receptors. Thus, the in vivoevaluation of EC72 also enabled evaluation of the extent ofkidney-specific damage caused by the systemic administration offolate-mitomycin conjugates.

Balb/c mice (5 mice/group) were given an intraperitoneal injection with1×10⁶ M109 cells. The mice were then given an intraperitoneal injectiononce daily with 1800 mmoles/kg of EC72 or mitomycin C (control mice wereinjected with PBS) beginning on day 5 after tumor cell innoculation(ILS=increased life span (i.e., median survival time)).

As shown in FIG. 2, all control mice died by day 22 post tumorinoculation. While a 39% increase in lifespan (ILS) was observed for theanimals treated with unmodified mitomycin C, the animals treated withEC72 had an average of a 178% increase in life span.

The same experiment (except that 30 daily injections with EC72 ormitomycin C were given) was repeated to confirm the observedEC72-mediated anti-tumor activity, and to confirm whether the effect ofEC72 was mediated by folate binding to its receptor. Thus, a group ofanimals were given intraperitoneal injections of EC72 plus a 10-foldexcess of free folic acid. As shown in FIG. 3, the group of animalstreated with EC72 showed the greatest increased life span (i.e., mediansurvival time; 185% over those of the PBS control group), while theanimals in the mitomycin C group showed only an approximate 25%increase. Furthermore, 1 of 4 EC72-treated animals emerged tumor-free byday 70 post tumor cell inoculation. The anti-tumor effect of the EC72conjugate was significantly reduced (112% increase in median survivaltime) by the coinjection of a moderate excess of folic acid, and allmice in this group eventually died from the tumor burden.

Major organs were also collected in both the EC72 and mitomycinC-treated animals (following euthanasia), and they were sent to anindependent pathologist for examination. As described in Table 2 below,the mitomycin C-treated animals suffered from extensivemyelosuppression, which is a characteristic side effect of mitomycin Ctherapy. In fact, all of the animals in this group died from apparentmitomycin C-related side effects. Surprisingly, animals treated withEC72 displayed no evidence of myelosuppression or kidney damage (i.e.,the spleen, femur bone marrow and kidneys all appeared normal to theexperienced pathologist). Interestingly, examination of blood collectedfrom EC72 treated animals indicated normal blood-urea-nitrogen andcreatine levels following 30 consecutive daily injections with EC72.Thus, folate-mitomycin C conjugates appear to be effectivechemotherapeutic agents that do not cause unwanted injury to normaltissues, including the folate receptor-positive kidneys.

TABLE 2 Test Article Dose Schedule/Route Duration Pathological FindingsDrug#1 1800 nmol/kg q1d, i.p. 21 days Bone (femur) was completelydepleted of marrow cells Abnormal spleen: loss of all spleen-derivedmarrow tissue Liver, kidney, heart, brain, lung, muscle and intestinevisually appeared normal No evidence of neoplasia in major organs EC721800 nmol/kg q1d, i.p. 44 days Bone marrow (femur) was normal Spleenappeared normal Kidney appeared normal Occasional neoplasia found inliver, lung and intestine; no evidence of hemorrhage or degenerationAnimals died from tumor burden

EXAMPLE 4 Survival of Tumor-Bearing Mice Injected Intravenously withEC72

The anti-tumor activity of EC72 when administered intravenously (i.v.)to tumor-bearing animals, was evaluated in Balb/c mice bearingsubcutaneous M109 tumors. Thus, a protocol similar to that described inExample 3 was followed except that the compounds were administered i.v.(described below) and the tumors were subcutaneous. Four days post tumorinoculation in the subcutis of the right axilla, mice were injected i.v.qd×5 for six weeks with 1800 nmoles/kg of EC72.

Tumor growth was determined at 2-day intervals in each treatment groupuntil the tumors grew to at least 1500 mm3. The tail veins becameexcessively bruised and inaccessible after 12 i.v. injections so dosingwas continued i.p. following the same qd×5 schedule. As shown in FIG. 4,EC72 treatment was effective in delaying the growth of the M109 tumor.Interestingly, the tumors did begin to increase their growth rateshortly after the switch was made to i.p. dosing. Thus, EC72 may nothave reached the distal subcutaneous tumor efficiently following i.p.administration of EC72, a limitation not encountered when EC72 wasadministered i.v. during the initial phase of the therapy.

EXAMPLE 5 Survival of Mice Bearing Solid Tumors Treated with EC72

The protocol described in Example 4 was followed except that mice wereinjected i.v. throughout the treatment period and were treated with EC72for 4 weeks. Balb/c mice (n=5) were injected subcutaneously in thesubcutis of the right axilla with 1×10⁶ M109 cells and were treated qd×5for 4 weeks with 1800 nmol/kg of EC72. EC72 was administered i.v.beginning on the designated day post tumor cell inoculation (PTI; seeTable 3). Animal survival was monitored daily, and mice showing acomplete response were those that were tumor-free at day 60 PTI.

In the subcutaneous tumor model, survival declined with the length indelay of EC72 administration. Thus, as summarized in Table 3,performance was maximal if treatment with EC72 began 1 day PTI, where 2of 5 complete responses resulted and 3 of 5 animals had a 133% increasein lifespan (ILS; i.e., partial response). In contrast, delaying EC72treatment until 12 days PTI resulted in only an 8% ILS with 0 of 5complete responses. Also, median survival time (days) and % ILSdecreased with an increasing delay in initiation of EC72 treatment.

TABLE 3 Animals/cohort n = 5 n = 5 n = 5 n = 5 n = 8 Initiation ofTreament PTI 1 PTI 3 PTI 7 PTI 12 Untreated Controls Median Survival(days) 56 54 28 26 24 % ILS 133 125 17 8 n/a Partial Responses 3 5 5 5n/a Complete Responses 2 0 0 0  0

EXAMPLE 6 Survival of Mice Bearing Solid Tumors Treated with EC72 andTAXOL®

The protocol described in Example 5 was followed except that animalswere injected i.v. at the beginning of the treatment period and wereinjected i.p. later in the treatment period. To investigate whether EC72and TAXOL® in combination might effectively increase survival of micebearing solid tumors, subcutaneous M109 tumors were formed in Balb/c(n=5 for each treatment group) mice for 12 days (i.e., a time PTI wheninitiation of treatment with EC72 alone was ineffective (see Table 3)).The mice were then treated with TAXOL® (i.e., paclitaxel) with orwithout EC72, and both primary tumor volumes and survival of the animalswere measured.

Twelve days PTI, the mice were treated with TAXOL® (20 mg/kg, i.v. ondays 12, 15, 19, 22, and 26 PTI) with or without EC72 (1800 nmol/kg qd×5for 4 weeks; i.v. on days 12, 15, 19, 22, and 26 PTI, and i.p. allremaining days). Tumor volumes were calculated using the equationV=a×b²/2, where “a” is the length of the tumor and “b” is the widthexpressed in millimeters. The tumors were measured using calipers.

As shown in FIG. 7, tumors in untreated control animals grew rapidly,and by day 50 PTI, they reached a size where euthanasia was required.Tumors in TAXOL®-treated animals failed to grow until day 29 PTI, afterwhich they resumed growth. Although treatment with 20 mg/kg of TAXOL®alone resulted in a 1.3 log cell kill (LCK), the animals treated withTAXOL® alone appeared sick, experienced weight loss, and none of the 5mice in the cohort were long term survivors. In contrast, tumors in micetreated with TAXOL® and EC72 in combination decreased in size during thedosing period, and this regimen resulted in a 1.8 LCK in 3 of the 5 micein this cohort, and 2 of 5 mice were tumor-free at 90 days PTI.Furthermore, all of the mice treated with TAXOL® in combination withEC72 maintained their weight and appeared healthy throughout the dosingperiod. These results show that EC72 and TAXOL® act synergistically toprevent tumor growth because EC72 alone failed to produce an anti-tumorresponse in this subcutaneous tumor model when EC72 treatment wasinitiated 12 days PTI (see Table 3), and the response with EC72 andTAXOL®D in combination was much greater than with TAXOL® alone.

EXAMPLE 7 Survival of Mice Bearing Solid Tumors Treated with EC72 andTAXOL®

The procedure described in Example 6 was followed except that TAXOL® wasdosed at 15 mg/kg and 6 mice/cohort were tested. As shown in FIG. 8,tumors in untreated control animals grew at an exponential rate, and byday 50 PTI, they reached a size where euthanasia was required. Incontrast, tumors in TAXOL®-treated mice failed to grow until day 21 PTI,after which they resumed apparent normal exponential growth. TAXOL®therapy resulted in a 0.6 LCK with a 135% increase in mean survival timein 4 of the 6 treated animals, and 2 of the 6 mice were completeresponders (see Table 4 where CR=complete response, PR=partial response,and NR=no response). As in Example 6, strikingly better results wereobtained with the TAXOL®-treated animals that were also treated withEC72. Thus, the combination of TAXOL® and EC72 produced a 1.5 LCK with a185% increase in mean survival time in 2 of 6 mice, and, importantly, 4of the 6 mice were complete responders (see FIG. 8 and Table 4). Takentogether, these results and the results shown in FIG. 7 demonstrate thatTAXOL® and EC72 act synergistically to inhibit tumor growth.

TABLE 4 Taxol Log Mean Dose EC72 Dose Cell Kill Survival Time (mg/kg)(nmol/kg) (LCK) (% T/C) CR PR NR 15 0 0.6 135 2 4 0 15 1800 1.5 187 4 20

The toxicity of each tested regimen was also monitored by measuringweight loss, blood chemistry, and tissue pathology. Animals treated withTAXOL® in combination with EC72 appeared healthy and did not lose weightthroughout the dosing period. Also, the tissue pathology results fromthe mice treated with TAXOL® and EC72 confirmed that there was no majororgan degeneration in these animals (Table 5).

TABLE 5 TAXOL ® 15 mg/kg, TAXOL ® 15 mg/kg + n = 6 mice EC72 n = 6 miceNormal Degeneration Normal Degeneration Heart 6 5 Liver 5 1(1) 4 2(1)Spleen 6 6 Kidney 6 5 1(2) Bone 6 6 “0.5” = Occasionally present, “1” =Mild, “2” = Moderate, “3” = Marked

EXAMPLE 8 Relative Affinity Assay

The affinity of EC72 for folate receptors (FRs) relative to folate wasdetermined according to a previously described method (Westerhof, G. R.,J. H. Schomagel, et al. (1995) Mol. Pharm. 48: 459-471) with slightmodification. Briefly, FR-positive KB cells were gently trypsinized in0.25% trypsin in phosphate-buffered saline (PBS) at room temperature for3 minutes and then diluted in folate-free RPMI (FFRPMI) containing 10%heat-inactivated fetal calf serum (HIFCS). Following a 5 min 800×g spinand one PBS wash, the final cell pellet was suspended in FFRPMI 1640 (noserum). Cells were incubated for 15 min on ice with 100 nM ³H-folic acidin the absence and presence of increasing concentrations of EC72 orfolate. Samples were centrifuged at 10,000×g for 5 min, and then thecell pellets were suspended in buffer, transferred to individual vialscontaining 5 mL of scintillation cocktail, and then counted forradioactivity. Negative control tubes contained only the ³H-folic acidin FFRPMI (no competitor). Positive control tubes contained a finalconcentration of 1 mM folic acid, and CPMs measured in these samples(representing non-specific binding of label) were subtracted from allsamples. Notably, relative affinities were defined as the inverse molarratio of compound required to displace 50% of ³H-folic acid bound to theFR on KB cells, and the relative affinity of folic acid for the FR wasset to 1. As shown in FIG. 9, EC72 has a relative affinity of 0.59,indicating that this conjugate can effectively compete with folic acid(the native ligand) for binding to the folate receptor.

EXAMPLE 9 In Vitro Dose Response

KB cells were seeded in 12-well Falcon plates and allowed to formnear-confluent monolayers overnight. Following one rinse with 1 mL offresh FFRPMI/HIFCS, each well received 0.5 mL of FFRPMI/HIFCS containingincreasing concentrations of EC72 in the presence of absence of 0.1 mMfolic acid (competitor). Cells were incubated for 15 min at 37° C. andthen rinsed four times with 0.5 mL of FFRPMI/HIFCS. 0.5 mL of freshFFRPMI/HIFCS was then added to each well, and cells were chased for atotal of 72 h at 37° C. Two hours before the end of the incubation,media was replaced in each well with 0.5 mL FFRPMI/HIFCS containing 5μCi of ³H-thymidine. Monolayers were then washed 3 times with 0.5 mL ofPBS and then precipitated with 1 ml of 10% cold trichloroacetic acid.The trichloroacetic acid solution was collected and discarded, andprecipitated material was sequentially dissolved in 0.5 mL 0.25 N NaOHand 1% sodium dodecyl sulfate for 15 min at room temperature. Sampleswere then counted for radioactivity using a Packard gamma counter. Finaltabulated values were expressed as a percentage of radioactivityincorporated into untreated cell samples. As shown in FIG. 10, EC72displays high, dose-responsive activity against FR-positive KB cells(IC₅₀ 5 nM), and the activity is specific since an excess of free folicacid effectively blocked any DNA synthesis inhibition.

EXAMPLE 10 In Vivo Dose Response

Six to seven week-old mice (female Balb/C strain) were obtained fromHarlan, Inc., Indianapolis, Ind. The mice were maintained on Harlan'sfolate-free chow for a total of three weeks prior to the onset of andduring this experiment. Folate receptor-positive M109 P₀ tumor cells(0.5×10⁶ cells per animal) were inoculated in the upper peritonealcavity 4 days prior to the onset of dosing. The formulations indicatedin FIG. 11 were dosed intraperitoneally qd×30 starting on day 4 posttumor inoculation, and animal survival was monitored daily. EC72displayed dose-dependent activity in M109 tumor-bearing mice. At the1800 nmol/kg dose level of EC72 (saturable to folate receptors in vivo),a 240% increase in lifespan was observed with 1 of 4 animals showing acomplete response.

EXAMPLE 11 In Vivo Activity on Folate Receptor-Negative Tumor

Six to seven week-old mice (female Balb/C strain) were obtained fromHarlan, Inc., Indianapolis, Ind. The mice were maintained on Harlan'sfolate-free chow for a total of three weeks prior to the onset of andduring this experiment. Folate receptor-negative 4T-1 tumor cells (1×106cells per animal) were inoculated in the subcutis of the right axilla.The formulations indicated in FIG. 12 were dosed intraperitoneally qd×30starting on day 4 post tumor inoculation, and tumor volume as well asanimal survival were monitored daily. EC72 had no activity against asubcutaneous folate-receptor-negative 4T-1 tumor.

1. A conjugate of the general formulaB-L-X wherein the group B is folate, or an analog or a derivativethereof, that binds to a surface accessible folate receptor that isuniquely expressed, overexpressed, or preferentially expressed by apopulation of pathogenic cells; wherein the group L comprises acleavable linker and wherein the cleavable linker is a disulfide group;and wherein the group X comprises a mitomycin compound, or an analog ora derivative thereof.
 2. The conjugate of claim 1 wherein the linker iscleaved under reducing conditions.
 3. The conjugate of claim 1 whereinthe mitomycin is selected from the group consisting of mitomycin A,mitomycin B, mitomycin C, mitomycin D, mitomycin E, mitomycin F, andporfiromycin.
 4. A method of selectively eliminating a population ofpathogenic cells in a host animal harboring said population of cellswherein the members of said cell population have a surface accessiblebinding site for folate, or an analog or derivative thereof, said methodcomprising the steps of: administering to said host a conjugate of thegeneral formulaB-L-X wherein the group B is folate, or an analog or a derivativethereof, that binds to a surface accessible folate receptor that isuniquely expressed, overexpressed, or preferentially expressed by thepopulation of pathogenic cells; wherein the group L comprises acleavable linker and wherein the cleavable linker is a disulfide group;and wherein the group X comprises a mitomycin compound, or an analog ora derivative thereof; and selectively eliminating said population ofpathogenic cells.
 5. The method of claim 4 wherein the linker is cleavedunder reducing conditions.
 6. The method of claim 4 wherein themitomycin is selected from the group consisting of mitomycin A,mitomycin B, mitomycin C, mitomycin D, mitomycin E, mitomycin F, andporfiromycin.
 7. The method of claim 4 wherein the population ofpathogenic cells is a cancer cell population.
 8. The method of claim 4further comprising the step of administering to said host a therapeuticfactor selected from the group consisting of a cell killing agent, atumor penetration enhancer, a chemotherapeutic agent, a cytotoxic immunecell, and a compound capable of stimulating an endogenous immuneresponse.
 9. The method of claim 8 wherein the therapeutic factor is achemotherapeutic agent.
 10. The method of claim 9 wherein thechemotherapeutic agent is paclitaxel.
 11. A pharmaceutical compositioncomprising a conjugate of the general formulaB-L-X wherein the group B is folate, or an analog or a derivativethereof, that binds to a surface accessible folate receptor that isuniquely expressed, overexpressed, or preferentially expressed by apopulation of pathogenic cells; wherein the group L comprises acleavable linker and wherein the cleavable linker is a disulfide group;and wherein the group X comprises a mitomycin compound, or an analog ora derivative thereof; and a pharmaceutically acceptable carriertherefor.
 12. The composition of claim 11 wherein the linker is cleavedunder reducing conditions.
 13. The composition of claim 11 wherein themitomycin is selected from the group consisting of mitomycin A,mitomycin B, mitomycin C, mitomycin D, mitomycin E, mitomycin F, andporfiromycin.
 14. The composition of claim 11 further comprising atherapeutic factor selected from the group consisting of a cell killingagent, a tumor penetration enhancer, a chemotherapeutic agent, and acompound capable of stimulating an endogenous immune response.
 15. Thecomposition of claim 14 wherein the therapeutic factor is achemotherapeutic agent.
 16. The composition of claim 15 wherein thechemotherapeutic agent is paclitaxel.
 17. A method of preparing abiologically active conjugate of the formulaB-L-X wherein B is folate or a folate-receptor-binding analog orderivative thereof; X comprises a mitomycin compound or an analog orderivative thereof; and L is a divalent linker comprising a disulfidebond, said method comprising the steps of forming a thiosulfonateintermediate of the formula B-(L″)nSSO2R or an intermediate of theformula X-(L′)nSSO2R and reacting said thiosulfonate intermediate with acompound of the formula X-(L′)n′-SH or B-(L″)n′-SH, respectively,wherein L′ and L″ are, independently, divalent linkers through which thethiol group SH is covalently bonded to B and X, respectively; n and n′are 1 or 0; and R is alkyl, substituted alkyl, aryl, heteroaryl orsubstituted aryl or heteroaryl.
 18. A conjugate comprising folate, or ananalog or derivative thereof, linked by a cleavable linker to amitomycin compound, or an analog or derivative thereof, wherein thecleavable linker is a disulfide group, wherein the folate binds to asurface accessible folate receptor that is uniquely expressed,overexpressed, or preferentially expressed by a population of pathogeniccells.
 19. A method of selectively eliminating a population ofpathogenic cells in a host animal harboring said population of cellswherein the members of said cell population have a surface accessiblebinding site for folate, or an analog or derivative thereof, said methodcomprising the steps of: administering to said host a conjugatecomprising folate, or an analog or derivative thereof, linked by acleavable linker to a mitomycin compound, or an analog or derivativethereof, wherein the cleavable linker is a disulfide group, wherein thefolate binds to a surface accessible folate receptor that is uniquelyexpressed, overexpressed, or preferentially expressed by a population ofpathogenic cells; and selectively eliminating said population ofpathogenic cells.